The DNA sequencing methods which have emerged since about 1970 have played a key role in the development of modern molecular biology and have enabled the investigation of such important problems as the molecular mechanism of life, the regulation and activity of genes, the localization of genes responsible for inherent characters and diseases, the structure of chromosomes, the disease-causing factors of bacteria and viruses, (e.g. HIV for AIDS), etc. Through sequencing techniques many useful and practical applications have developed, i.e. genetic diseases can be diagnosed, certain sequence locations are now used by law enforcement agencies as `finger prints` of individuals, genetic research in agriculture is speeded up using specific DNA sequences.
One technique for DNA sequencing referred to as the Gilbert method, [Gilbert et al., Proc. Natl. Acad. Sci., 74, 560, (1977), and Gilbert, Science, 214, 1305 (1981)] and is based on a chemical cleavage process which breaks a 5' end .sup.32 P-labeled DNA at specific bases. Using purine-specific dimethylsulphate and pyrimidine-specific hydrazine, four chemical reactions yield DNA fractions according to their end base grouping. The products are then resolved by size, using polyacrylamide gel electrophoresis and the pattern of bands are read via autoradiography.
The Sanger method [Sanger et al, Proc. Natl. Acad. Sci., 74, 5464, (1977)] sequences single stranded DNA by an enzymatic chain-terminating method. A preliminary .sup.32 P-labeled primer is incubated with the DNA template to be analyzed in the presence of DNA polymerase and a proper mixture of `regular` deoxy- and `chain terminatory` dideoxy-ribonucleoside triphosphates. The template is copied by the appropriate nucleotides (A, G, C or T) into the complementary strand which grows from the 3' end of the template until a chain terminator is built in. If the terminator is a ddTTP, all copies of the template will end in a T (thymine base). The procedure is repeated with A (adenine), G (guanine) and C (cytosine) terminators respectively. These segments for each procedure (A, G, C or T) are resolved by gel electrophoresis and read via autoradiography.
The Sanger chain terminating method is currently the most popular DNA sequencing procedure since it is easy to read and easy to automate However, both the Sanger and Gilbert methods have serious drawbacks. The manual procedures are repetitive, laborious and time consuming. The DNA sequencing of some gene region in preparation for an actual research problem often takes several years. When one considers that an individual gene often consists of several hundred thousand base pairs, that a chromosome often consists of thousands of genes and a human genome consists of 46 chromosomes, the numbers of steps in sequencing of a human genome becomes astronomical. It is stated that the number of base pairs in a human genome is over three billion. Molecular biologists also have strong interests in mapping the sequence of genes from other species in both plant and animal life including, but not limited to, mice, yeasts, bacteria and viruses.
In addition to slow manual procedures, there is also the problem of using radioisotopes for labeling. They are hazardous to health, expensive, unstable for storage and difficult to dispose of in an environmentally sound way.
It has been proposed that the limitations of using radioisotopes might be overcome by use of fluorescent dye labeling followed by gel electrophoresis. Various methods have been proposed using fluorophores such as fluorescein iodoacetamide, succinyl fluorescein derivatives. It has also been proposed to use four different colored fluorophores each being specific of each of the four bases contained in DNA. As attractive at these concepts may be they also have inherent drawbacks in that only a single set of sequence data can be obtained from each gel electrophoresis. Gel electrophoresis is one of the most laborious and time-consuming steps.
Southern [J. Molecular Biol., 98, 503 (1975)] developed a technique for blotting DNA from a gel onto a cellulose nitrate membrane in much the same manner as using an ink blotter. An improvement of this technique was made by Church and Gilbert [Proc. Natl. Acad. Sci., 81, 1991 (1988)] who applied an electric field to transfer unlabeled DNA fragments onto a nylon membrane followed by a subsequent ultraviolet-crosslinking step to bind the DNA covalently to the nylon. A hybridization step with short .sup.32 P-labeled single strand oligonucleotide produced the image of the DNA sequence ladder. This method is a significant advance over other methods in that numerous different sequences can be mixed, loaded and separated on a single sequencing gel, followed by a transfer to a nylon membrane and probed many times using different complementary probes and producing separate autoradiographs for each sequence in the cycle. This method, referred to as `multiplexing` [Church et al., Science, 240, 185 (1988)] still requires the expense and time consuming work of producing and handling a large number of autoradiographs. Nylon membranes are not suited for use with DNA segments tagged with fluorophores because polyamides exhibit an unacceptably high background fluorescence.
A method of sequencing DNA using fluorophores may be found, for example, in Middendorf, U.S. Pat. No. 4,729,947 where segmented DNA strands are marked at one end with biotin. Using a continuous method of electrophoresis, they are moved into avidin marked with fluorescein. Avidin has a high affinity for biotin. The shorter strands, being resolved first, combine with the avidin and are scanned and the signals decoded and the process continues as the longer strands are resolved. There is no use of blotting membranes or materials.
Another method is found in Van den Engh et al, U.S. Pat. No. 4,770,992 wherein chromatin (comprising DNA and protein) is first contacted with a cross-linking agent for the protein to provide a substantially rigid chromatin particle. The DNA is then separated into individual DNA strands and contacted with a complementary polynucleotide probe specific for the DNA sequence of interest. The probe is marked with a fluorescent label. The fluorophore tagged DNA sequences are then detected by subjecting the probe to a suitable light source using flow cytometry and detecting the light emitted by the fluorescent label so as to identify the preselected DNA sequence. However, this method does not provide a means for stripping the probe and reprobing the sample using different DNA sequences as can be accomplished on an appropriately designed membrane.